Patent RU-2703282-C1 (RU)
Title: Method for producing a diagnosticum for detecting a toxin of a cholera vibrio recovered from environmental objects
Inventor: Pisanov Ruslan Vyacheslavovich
IPC: A61K39/106
Filed: 2019-01-10
Granted: 2019-10-16
Abstract: FIELD: medicine. 
     SUBSTANCE: invention refers to medicine, namely to microbiology, and can be used to produce a diagnosticum for quantitative determination of toxin of cholera vibrio recovered from environmental objects. That is ensured by producing experimental antitoxic immune rabbit serum by immunizing rabbits weighing 2.5–3.0 kg by administering a pure cholera toxin preparation in dose of 100 mcg of protein subcutaneously into the inguinal lymph node and intravenously into a marginal ear vein in amount of 3 injections with interval of 14 days, then 14 days after the last injection, rabbits are bleeding, and blood is prepared with serums to determine antibody titre. Further, immunoglobulins are recovered from rabbit serums, for this purpose physiological saline is prepared in advance, buffered with disubstituted sodium phosphate and monosubstituted potassium phosphate at pH 7.2 (PBS), then preparing an aqueous solution of methanol, mixing 6 ml of methanol with 8 ml of distilled water, the mixture is cooled, after which the obtained serum in amount of 4 ml is combined with 2 ml of buffered saline (PBS), placed in centrifuge cups, put on ice and poured into them whey at temperature 0 °C, then buffered physiological solution is added, mixed, methanol water is poured in with 14 ml, while reducing the temperature to -5° C, after which sleeve with mixture is put in refrigerator for 30–40 minutes, and then centrifuged at 2,000 rpm for 20 minutes at zero temperature, obtained supernatant is drained, and the precipitate is suspended in ½ PBS from the initial volume of whey is determined by the method of Lowry, resulting in sensitin. Polymer support is prepared using an anionic polymerization method of a monomer – acrylic aldehyde – in an aqueous-alkaline medium to obtain monodisperse particles of a spherical shape with diameter of 1.0±0.1 mcm, which are stained with thianine and washed with distilled water. Polymeric carrier is then sensitized with immunoglobulins to the cholera toxin; for this purpose, the deposited carrier residue is suspended in a borate buffer (pH 8.4), immunoglobulins are added thereto and allowed to contact while stirring for 2 hours at 20 °C, as a result, aldehyde reaction groups on the surface of particles specifically interact with amino groups of protein molecules of immunoglobulins. After that, suspension is cooled to 4–5 °C and held at this temperature for 16–18 hours. Free aldehyde groups are blocked by adding 0.5 % solution of gelatase to the suspension in buffered saline (pH 7.0–7.1) and incubation for 2 hours at room temperature. Suspension of diagnosticum is centrifuged for 10 minutes at 6,000 rpm and washed three times with buffered physiological solution (pH 7.0–7.1) from excess gelatine, and final sediment is suspended in 0.1 % solution of gelatase in buffered physiologic saline (pH 7.0–7.1). Diagnosticum is lyophilized by suspending preparation in 20 ml of 3 % gelatose-sucrose medium, then the suspension is poured into ampoules of 1 ml, frozen in liquid nitrogen with subsequent vacuum drying, gradually raising temperature to 22–23 °C for a day. 
     EFFECT: use of the given method enables producing a diagnosticum for quantitative determination of cholera vibrio toxin, recovered from environmental objects, in order to detect epidemic dangerous toxigenic strains of cholera vibrio. 
     3 cl, 3 tbl, 2 ex